INVERSE PCR TO PERFORM LONG-DISTANCE HAPLOTYPING: MAIN APPLICATIONS TO IMPROVE PREIMPLANTATION GENETIC DIAGNOSIS IN HEMOPHILIA

MARCHIONE, VANINA DANIELA; NEME, DANIELA; DE BRASI, CARLOS DANIEL; ABELLEYRO, MIGUEL MARTÍN; RADIC, CLAUDIA PAMELA; MEDINA-ACOSTA, ENRIQUE; PALMITELLI, MICAELA; LARRIPA, IRENE BEATRIZ; ROSSETTI, LILIANA CARMEN

EUROPEAN JOURNAL OF HUMAN GENETICS

NATURE PUBLISHING GROUP

Lugar: Londres; Año: 2019 vol. 27 p. 603 - 611

1018-4813

Among other applications of long-distance haplotype phasing in clinical genetics, determination of linked DNA markers assurrogate for problematic structural variants (e.g., repeat-mediated rearrangements) is essential to perform diagnosis fromlow-quality DNA samples. We describe a next-of-kin-independent (physical) phasing approach based on inverse-PCR(iPCR) paired-end amplification (PI). This method enables typing the multialleles of the short tandem repeat (STR) F8Int21[CA]n at the F8-intron 21, as a surrogate DNA marker for the F8-intron 22 inversion (Inv22), the hemophilia A-causativehotspot, within the transmitted haplotype in informative carriers. We provide proof-of-concept by blindly validating the PIapproach in 15 carrier mother/affected-son duos. Every F8Int21[CA]n STR allele determined in phase with the Inv22 allele in the female carriers from the informative duos was confirmed in the hemizygous proband (P = 0.00003). A secondsurrogate STR locus at the F8-IVS22 was obtained by the PI approach improving severe-HA preimplantation genetic diagnosis by augmenting heterozygosity in Inv22 carriers bypassing the requirement for family linkage analysis. The ability of the PI-assay to combine other marker pairs was demonstrated by haplotyping a SNV (F8:c.6118T > C) with a >28kbdistant F8-IVS22 STR. The PI approach has proven flexibility to target different marker pairs and has potential for multiplex characterization of iPCR products by massively parallel sequencing.